This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The goal of this project is to further understand how cells communicate during the formation of the blastema in a regenerating limb. The process of limb regeneration requires cells to interact that do not normally interact -such as when epithelial cells on the dorsal and ventral sides of a wound interact during wound closure. Upon excision and healing of parts of the blastema these cells are capable of sensing that their new neighbor is different from their normal neighbor, despite the lack of obvious differences among these cells. Not surprisingly, cell-cell interactions are essential for communication of cell identity. Yet, exactly how these cells are different, and how the signal of cell identity is transmitted among cells remains a mystery. I hypothesize that the blastema undergoes metabolic oscillations, possibly utilizing secondary messengers (such as Ca++) to transmit this signal through cell-cell junctions. Thus when two cells interact that do not normally interact, they are metabolically out of phase and recognize each other as "different". I propose to utilize a calcium biosensor (called XXL) and determine whether synchronized calcium oscillations occur periodically in the blastema. This biosensor contains a short calcium-binding peptide between CFP and YFP. Upon calcium binding the short peptide changes conformation, which brings the CFP and YFP molecules in close proximity and allows exited CFP to FRET to YFP. FLIM will be used to measure oscillations in FRET in tissue culture cells, axolotl blastema tissue explants, and eventually in vivo blastemas. It is possible that a different secondary messenger such as cAMP or IP3 mediates metabolic oscillations occurring in the blastema. In this case, similar biosensors can also be utilized for these molecules.